RANK1, an ankyrin-repeat containing peptide from rice associated with disease resistance

ABSTRACT

An isolated nucleic acid comprising a sequence of SEQ ID NO:2. The nucleic acid sequence can be transformed into plant cells. The nucleic acid codes for disease resistance in plants. The transformed plant cells can then be introduced to plants for regeneration of disease resistant plants.

[0001] This application is a continuation of application Ser. No.09/525,223, which is a continuation-in-part of PCT internationalapplication No. PCT/SG97/00042 which has an international filing date ofSep. 15, 1997, which designated the United States, the entire contentsof which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] Fungal, bacterial and viral diseases in crop plants result inreduced yields and product quality and are responsible for substantiallosses to farmers. For example, rice blast, an often devastating diseasethat occurs in most rice growing areas worldwide, is estimated to costfarmers $5 billion a year (Moffat, 1994). The disease reduces rice yieldsignificantly, particularly in the temperate flooded and tropical uplandrice ecosystems. The use of resistant cultivars is the most economicaland effective method of controlling the disease. Over the last decades,much has been learned about the genetics of resistance to the blastfungus. Many major genes for resistance have been identified and widelyused in breeding programs. However, the molecular mechanism of hostresistance to this pathogen is mostly unknown.

[0003] When a plant is attacked by a pathogen such as the rice blastfungus, it can in most cases fend off the infection by mounting abattery of defense responses (Lindsay et al., 1993). The activation ofplant defense occurs upon pathogen recognition and results in the haltof pathogen ingress. Systemic acquired resistance (SAR) is one importantcomponent of this complex system that plants use to defend themselvesagainst pathogens (Ryals et al., 1996). SAR can be triggered by a localhypersensitive response(HR) to an avirulent pathogen, which rendersuninfected parts of the plant resistant to a variety of normallyvirulent pathogens. SAR is a particularly important aspect ofplant-pathogen response because it is a pathogen inducible, systemicresistance against a broad spectrum of pathogens.

[0004] Significant progress has been made recently in decipheringmolecular aspects of SAR. The Arabidopsis gene NPR1/NIM1 has been clonedusing a map-based strategy (Cao et al., 1997; Ryals et al., 1997).Mutants with defects in NPRI/NIMl fail to respond to variousSAR-inducing treatments, displaying little expression ofpathogenesis-related (PR) genes and exhibiting increased susceptibilityto infections. The gene encodes a novel protein containing ankyrinrepeats and shows homology to the mammalian signal transduction factorIκB subclass a, suggesting that RPN1/NIM1 may interact with anNF-κB-related transcription factor to induce SAR gene expression andtrigger disease resistance (Ryals et al., 1997).

[0005] The ankyrin repeat is a 33-amino acid motif present in a numberof proteins of diverse functions including transcription factors, celldifferentiation molecules, and structural proteins (Bennet, 1993). Theankyrin motif consensus sequence contains the following sequence ofamino acids shown as SEQ ID NO:1:

-D----G-TPLH-AA-------V--LL--GA-

[0006] (LaMarco, 1991). This motif has been shown to mediate proteininteractions and is usually present in tandem arrays of four to sevencopies (Michaely and Bennett, 1993). Ankyrin repeat-containing proteinshave been shown to have diverse functions and to be involved inprotein-protein interactions. Some of these proteins in mammals aretranscription-regulating proteins, such as the NF-κB, inhibitor IκB(Baldwin, A. 1996; Whiteside et al., 1997). The NF-κB/IκB signaltransduction pathways are conserved in both mammals and flies. Astimulus such as IL-1 treatment or bacterial inoculation leads toactivation of a signal transduction pathway because of the degradationof IκB or its homolog and the release of the NF-κB transcription factorto the nucleus to stimulate transcription (Baeuerie and Baltimore, 1996;Baldwin, 1996). In Arabidopsis, NPR1/NIM₁, which is homologous to theNF-κB inhibitor IκB, controls the onset of SAR. The transcription factortargeted by NPR1/NIM1 serves as a repressor of SAR gene expression anddisease resistance either by direct or indirect action (Ryals et al.,1997).

[0007] SAR is an important plant defense mechanism against infectiouspathogens. For example, evidence suggests that SAR can protect plantsagainst rice blast disease. The SAR inducer benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (“BHT”) was foundto be effective in controlling the blast disease in field conditions.

SUMMARY OF THE INVENTION

[0008] A gene has been isolated from blast resistant plants that encodesa novel protein containing ankyrin repeats. This gene, designated RANK1,for rice ankyrin repeats, has significant homology to the Arabidopsisgene NPR1/NIM1 and the mammalian signal transduction factor inhibitorI-κB. The RANK1 gene encodes a protein that is believed to play animportant role in rice defense to the blast pathogen infection as wellas to other diseases which respond through SAR. Since both the RPN1/NIM1and RANK1 genes code for ankyrin repeats, it is believed that theserepeats may be responsible for SAR induced resistance to plant disease,especially rice blast disease.

[0009] Accordingly, the present invention provides, in one embodimentthereof, an isolated nucleic acid comprising a sequence of SEQ ID NO:2.

[0010] In another embodiment, the invention provides recombinant DNAexpression vectors functional in a plant cell comprising a nucleic acidof SEQ ID NO:2.

[0011] A third embodiment is a plant cell stably transformed with anucleic acid comprising a sequence of SEQ ID NO:2.

[0012] Yet another embodiment provides a transgenic plant transformedwith a nucleic acid comprising a sequence of SEQ ID NO:2.

[0013] The invention further provides a method of conferring resistanceto disease in a monocotyledonous plant comprising stably integratinginto the genome of said plant the nucleic acid having the sequence whichcodes for a protein comprising the ankyrin motif sequence.

[0014] Another embodiment of the invention provides a method ofconferring resistance to rice blast disease in a monocotyledonous plantcomprising stably integrating into the genome of said plant the nucleicacid having the sequence which codes for a protein comprising theankyrin motif sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 Shows the alignment of RANK1's predicted amino acidsequence (SEQ ID NO:3) with Iκβ-E (SEQ ID NO:4), Iκβ-α (SEQ ID NO:5),Cactus (SEQ ID NO:6) and NPR1 (SEQ ID NO:7) proteins containing ankyrinrepeats.

[0016]FIG. 2 Agarose gel electrophoresis showing accumulation of NPR1RNA in blast resistant plants post-inoculation. RANK1 specific primerswere used to amplify cDNAs isolated from the resistant (C101A51) andsusceptible (CO039) plants.

[0017]FIG. 3 Shows alignment of the RANK1 partial cDNA (SEQ ID NO:2) andgenomic DNA (SEQ ID NO:8).

[0018]FIG. 4 Shows the Southern analysis of the resistant (C101A51) andsusceptible (Co39) plants with the RANK1 gene.

[0019]FIG. 5 Shows the Northern analysis of the resistant (C101A51) andsusceptible (Co39) plants with the RANK1 gene.

[0020]FIG. 6 Shows the full-length cDNA of RANK1 gene (SEQ ID NO:9)which obtained from rice line C101A51 by 5′ RACE (5′ Rapid Amplificationof cDNA Ends), RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction)and screening of a cDNA library. The full-length cDNA of RANK1 is 2127bp of which the nucleotides from 1 to 999 were obtained by 5′ RACE andthe nucleotides from 798 to 2127 were inferred from a cDNA clone. The 5′RACE product and the cDNA clone have an overlapping region of 202 bpcorresponding to nucleotide position from 798 to 999. The ORF (OpenReading Frame) to RANK1 corresponds to nucleotide position from 808 to1830 (including the stop code “TAG”) which encodes a protein of 340amino acids.

[0021] The partial cDNA (SEQ ID NO:2), is a total of 573 bp whichcorresponds to nucleotide position from 1311 to 1883 on the full-lengthcDNA of RANK1 gene.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention relates to an isolated nucleic acidconferring resistance to rice blast disease. The nucleic acid codes fora protein which is predicted to have ankyrin repeats. The nucleic acidadvantageously has nucleotide sequence SEQ ID NO:2. It will berecognized, however, that the nucleotide sequence may vary, as permittedby the degeneracy of the genetic code while still encoding the sameprotein. Expression of the gene in a plant may be enhanced by replacingone or more of the codons shown in SEQ ID NO:2 with codons that arepreferred by the plant into which the nucleic acid is inserted.

[0023] The nucleic acid can be incorporated in plant or bacterium cellsusing conventional recombinant DNA technologies. Generally, suchtechniques involve inserting the nucleic acid into a DNA expressionvector. Such vector advantageously contains the necessary elements forthe transcription and translation of the inserted protein codingsequences and one or more marker sequences to facilitate selection oftransformed cells or plants.

[0024] A number of plant-active promoters are known in the art and maybe used to effect expression of the nucleic acid sequences disclosedherein. Suitable promoters include, for example, the nos promoter, thesmall subunit chlorophyll A/B binding polypeptide, the 35S promoter ofcauliflower mosaic virus, and promoters isolated from plant genes. Thepromoter may be isolated from the type of plant to be transformed. The35S or actin promoters may also be used for isolated cDNA clones. Theseare also useful to test overexpression of the gene.

[0025] Once the nucleic acid of the present invention has been clonedinto an expression vector, it is ready to be transformed into a plantcell. The term plant cell includes any cell derived from a plant; thisincludes undifferentiated tissues such as callus and suspensioncultures, as well as plant seeds, pollen or plant embryos. Plant tissuessuitable transformation include leaf tissues, root tissues, meristems,protoplasts, hypocotyls cotyledons, scutellum, shoot apex, root,immature embryo, pollen, and anther.

[0026] One technique of transforming plants with the nucleic acidconferring disease resistance in accordance with the present inventionis by contacting tissue of such plants with an inoculum of a bacteriatransformed with a vector comprising a nucleic acid in accordance withthe present invention. Generally, this procedure involves inoculatingthe plant tissue with a suspension of bacteria and incubating the tissuefor 48 to 72 hours on regeneration medium without antibiotics at250-280°C.

[0027] Bacteria from the genus Agrobacterium can be utilized totransform plant cells. Suitable species of such bacterium includeAgrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacteriumtumefaciens (eg., strains LBA4404 or EHA105) is particularly useful dueto its well-known ability to transform plants.

[0028] Another approach to transforming plant cells with the nucleicacid of this invention involves propelling inert or biologically activeparticles at plant cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006 and 5,100,792 all to Sanford et. al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the nucleic acid conferring diseaseresistance. Biologically active particles (e.g., dried yeast cells,dried bacterium or a bacteriophage, each containing DNA sought to beintroduced) can also be propelled into a plant cell tissue.

[0029] Another method of transforming plant cells is the electroporationmethod. This method involves mixing the protoplasts and the desirednucleic acid and forming holes in the cell membranes by electric pulseso as to introduce the DNA in the cells, thereby transforming the cells.This method currently has high reproducibility and various genes havebeen introduced into monocotyledons, especially rice plants by thismethod (Toriyama et. al., (1988); Shimamoto et al., (1989; Rhodes etal., (1988)).

[0030] Similar to the electroporation method is a method in which thedesired gene and protoplasts are mixed and the mixture is treated withPEG, thereby introducing the gene into the protoplasts. This method isdifferent from the electroporation method in that polyethylene glycol(“PEG”) is used instead of the electric pulse. (Zhang W. et. al.,(1988); Datta et al. (1990; Christou et al. (1991).

[0031] Other methods include 1) culturing seeds or embryos with nucleicacids (Topfer R. et al., (1989)); Ledoux et al., (1974); 2) treatment ofpollen tube, (Luo et al. (1988)); 3) liposome method (Caboche (1990)) ;Gad et al.(1990); and 4) the microinjection method (Neuhaus et al.(1987).

[0032] Known methods for regenerating plants from transformed plantcells may be used in preparing transgenic plants of the presentinvention. Generally, explants, callus tissues or suspension culturescan be exposed to the appropriate chemical environment (e.g., cytokininand auxin) so the newly grown cells can differentiate and give rise toembryos which then regenerate into roots and shoots.

[0033] The nucleic acid sequence of the present invention can be used toconfer to monocotyledonous plants, resistance to rice blast disease andother diseases regulated by SAR. Such plants include but are not limitedto rice, wheat, barley, maize and asparagus.

[0034] The invention is further illustrated by the following examples,which are intended to be illustrative and not to be limiting.

EXAMPLES Materials and Methods

[0035] Rice Plants and Blast Inoculation:

[0036] The resistant isogenic line C1o1A51 carrying the Pi-2 gene andthe susceptible cultivar CO39 were used in the experiment. Three-weekold rice plants were inoculated with isolate PO6-6 and kept in a dewchamber for 24 hours at 26 C. Leaf tissue was harvested from bothcultivars at 0, 4, 8, 12, 24, 48, 72 hours after inoculation.

[0037] RNA Isolation and RT-PCR

[0038] RNeasy mini kit (Qiagen, USA) was used to isolate total RNA from150-200 mg rice leaf tissue. Poly(A)+RNA fractionated from total RNAusing Qiagen Oligotex Spin Column, was used as a template in a reversetranscriptase-mediated polymerase chain reaction (RT-PCR) using 10-merrandom primers (Operon Technology, Inc). RT-PCR was conducted followingprotocols provided by the manufacturer (GIBCO-BRL, USA). The amplifiedcDNAs were then separated in 4.5% sequencing gel.

[0039] Cloning and DNA Sequencing

[0040] Specific bands were cloned into pGEM-T vector (Promega, USA).Clones were sequenced using the ABI PRISM 377 DNA sequencer(Perkin-Elmer, Calif., USA). The sequence was analyzed with softwaresDNAstar and Sequencer 3.0.

Results

[0041] RANK1 was Strongly Induced in the Resistant Plants

[0042] Twenty-eight random primers have been used to amplify cDNAs fromC1O1A51 and CO39. When primer OPF-1 (ACGGATCCTG; SEQ ID NO:10) was usedin the RT-PCR reaction, a specific band (about 600 bp) was observed onlyin the inoculated resistant plants. It was strongly induced as early as4 hours post-inoculation. This band was cut from the sequencing gel,re-amplified using the same primer and cloned into the PGEM-T vector.The DNA sequence of this cDNA clone is provided in SEQ ID NO:2. It wascompared to databases of known genes to search for homology to knowngenes. The search revealed that the predicted amino acid sequence of theprotein encoded by this gene (RANK1) has significant homology to thoseproteins containing ankyrin repeats including the Arabidopsis geneRPN1/NIM1 and mammalian gene family Iκβ (FIG. 1).

[0043] A pair of RANK1 specific primers was designed and used to amplifycDNAs isolated from the second inoculation experiment. Amplified cDNAswere run on agarose gel. The 600 bp fragment was only observed in theresistant plants (FIG. 2).

[0044] A Southern and Northern Analysis of the resistant (C101A5) andsusceptible (CO39) plants were performed as shown in FIGS. 4 and 5.

[0045] Isolation of the RANK1 Genomic Clone from a Bacterial ArtificialChromosome (BAC) Library.

[0046] The RANK1 partial cDNA clone was used as the probe to screen aBAC library made from an indica cultivar IR64. Six positive BAC cloneswere identified and minipreped for further subcloning. The sequence of a2.0 kb subclone revealed the presence of introns in the region spannedby the 600 bp cDNA fragment, designating the RANK1 gene. The sequence ofthe RANK1 genomic clone is set forth in SEQ ID No.:8.

BIBLIOGRAPHY

[0047] Baeuerie (1996) Cell 87:13-20.

[0048] Baldwin (1996) Annu. Rev. Immunol. 14:649-681.

[0049] Bennet (1993) J. Biol. Chem. 22703-22709.

[0050] Caboche et al. (1990) Physiol. Plant. 79:173-176.

[0051] Cao et al. (1997) Cell 88:57-63.

[0052] Christou et al. (1991) Bio/Technology 9:957-962.

[0053] Datta et al. (1990) Bio/Technology 8:736-740.

[0054] Gad et al. (1990) Physiologia Plantarium 79:177-183.

[0055] Gorlach et al. (1996) The Plant Cell 8:629-643.

[0056] LaMarco et al. (1991) Science 253:789-792.

[0057] Luo et al. (1988) Plant Molecular Biology Reporter 6(3):165-174.

[0058] Maniatis et al., Molecular Cloning: A Laboratory Manual (1982).

[0059] Moffat (1994) Science 265:1804-1805.

[0060] Neuhaus et al. (1987) Theoretical and Applied Genetics 75:30-36.

[0061] Rhodes et al. (1988) Science 240:204-207.

[0062] Ryals et al. (1997) The Plant Cell 9: 425-439.

[0063] Shimamoto et al. (1989) Nature 338:274-277.

[0064] Topfer et al. (1989) The Plant Cell 1:133-139.

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[0066] Whiteside et al. (1997) The EMBO Journal 16:1413-1426.

[0067] Zhang et al. (1988) Theoretical and Applied Genetics 76:835-840.

1 10 1 32 PRT Arabidopsis sp. misc_feature (1)..(1) Xaa = any amino acid1 Xaa Asp Xaa Xaa Xaa Xaa Gly Xaa Thr Pro Leu His Xaa Ala Ala Xaa 1 5 1015 Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Leu Leu Xaa Xaa Gly Ala Xaa 20 2530 2 573 DNA Artificial Sequence misc_feature (1)..(573) Oryza sp. RANK1cDNA 2 tacggatcct gctgcactga taaagtactg gaatgaccca gaaacatttc gaaagatcag60 ccaggcaatg gggcctttag gcggccctga ttttgctgaa ccttctggaa ctgaaggaac 120agaggaagaa ggtgaatatg aagatgaatc tatcgtccat cacactgcca gtgttggtga 180tgatgagggt ctgaagaagg ctttagatgg tggagcagac aaagacgaag aagacttgga 240gggcagaagg gccttacact ttgtatgtgg atacggggag ttgaagtgtg cccaagtact 300tcttgaggcg ggtgctgcag tggatgcttt ggacaagaac aagaacactc cgctgcatta 360cgccgctggc tatggtatga aggggtgcgt ggatcttctg ctgaagaacg gagccgctgt 420caccctcgaa aacatggatg gcaagacgcc cattgacgtt gcgaagctca acaaccagga 480tgaggttctc aagttgctgg aaaaggatgc cttcctgtag atcgcctttg ttattctcat 540gggcgcatga acagtttggc tccaggatcc gta 573 3 172 PRT Oryza sp.misc_feature (1)..(172) RANK1 predicted amino acid sequence 3 Thr AspPro Ala Ala Leu Ile Lys Tyr Trp Asn Asp Pro Glu Thr Phe 1 5 10 15 ArgLys Ile Ser Gln Ala Met Gly Pro Leu Gly Gly Pro Asp Phe Ala 20 25 30 GluPro Ser Gly Thr Glu Gly Thr Glu Glu Glu Gly Glu Tyr Glu Asp 35 40 45 GluSer Ile Val His His Thr Ala Ser Val Gly Asp Asp Glu Gly Leu 50 55 60 LysLys Ala Leu Asp Gly Gly Ala Asp Lys Asp Glu Glu Asp Leu Glu 65 70 75 80Gly Arg Arg Ala Leu His Phe Val Cys Gly Tyr Gly Glu Leu Lys Cys 85 90 95Ala Gln Val Leu Leu Glu Ala Gly Ala Ala Val Asp Ala Leu Asp Lys 100 105110 Asn Lys Asn Thr Pro Leu His Tyr Ala Ala Gly Tyr Gly Met Lys Gly 115120 125 Cys Val Asp Leu Leu Leu Lys Asn Gly Ala Ala Val Thr Leu Glu Asn130 135 140 Met Asp Gly Lys Thr Pro Ile Asp Val Ala Lys Leu Asn Asn GlnAsp 145 150 155 160 Glu Val Leu Lys Leu Leu Glu Lys Asp Ala Phe Leu 165170 4 198 PRT mammalian misc_feature (1)..(198) Ik beta-E 4 Asp Gly AspThr Leu Val His Leu Ala Val Ile His Glu Ala Pro Ala 1 5 10 15 Val LeuLeu Cys Cys Leu Ala Leu Leu Pro Gln Glu Val Leu Asp Ile 20 25 30 Gln LeuTyr Gln Thr Ala Leu His Leu Ala Val His Leu Asp Gln Pro 35 40 45 Gly AlaVal Arg Ala Leu Val Leu Lys Gly Ala Ser Arg Ala Leu Gln 50 55 60 Asp ArgHis Gly Asp Thr Ala Leu His Val Ala Cys Gln Arg Gln Ser 65 70 75 80 TrpPro Val Pro Ala Ala Cys Trp Lys Gly Gly Pro Glu Pro Gly Arg 85 90 95 GlyThr Ser Gln Gly Leu Ala Cys Leu His Ile Ala Thr Leu Gln Lys 100 105 110Asn Gln Pro Leu Met Glu Leu Leu Leu Arg Asn Gly Ala Asp Ile Asp 115 120125 Val Gln Glu Gly Ser Gly Lys Thr Ala Leu His Leu Ala Val Glu Thr 130135 140 Gln Glu Arg Gly Leu Val Gln Phe Leu Leu Gln Ala Gly Ala Gln Val145 150 155 160 Asp Ala Arg Met Leu Asn Gly Cys Thr Pro Leu His Leu AlaAla Gly 165 170 175 Arg Gly Leu Met Gly Ile Ser Ser Thr Leu Cys Lys AlaGly Ala Asp 180 185 190 Ser Leu Leu Arg Asn Val 195 5 165 PRT mammalianmisc_feature (1)..(165) Ik beta-a 5 Asp Gly Asp Ser Phe Leu His Leu AlaIle Ile His Glu Glu Lys Ala 1 5 10 15 Leu Thr Met Glu Val Ile Arg GlnVal Lys Gly Asp Leu Ala Phe Leu 20 25 30 Asn Leu Gln Gln Thr Pro Leu HisLeu Ala Val Ile Thr Asn Gln Pro 35 40 45 Glu Ile Ala Glu Ala Leu Leu GlyAla Gly Cys Asp Pro Glu Leu Arg 50 55 60 Asp Phe Arg Gly Asn Thr Pro LeuHis Leu Ala Cys Phe Gln Gly Cys 65 70 75 80 Leu Ala Ser Val Gly Val LeuThr Gln Ser Cys Thr Thr Pro His Leu 85 90 95 His Ser Ile Asn Gly His ThrCys Leu His Leu Ala Ser Ile His Gly 100 105 110 Tyr Leu Gly Ile Val GluLeu Leu Val Ser Leu Gly Ala Asp Val Asn 115 120 125 Ala Gln Glu Pro AsnGly Arg Thr Ala Leu His Leu Ala Val Asp Leu 130 135 140 Gln Asn Pro AspLeu Val Ser Leu Leu Leu Lys Cys Gly Ala Asp Val 145 150 155 160 Asn ArgVal Thr Tyr 165 6 198 PRT Cactus 6 Asp Gly Asp Thr Pro Leu His Leu AlaCys Ile Ser Gly Ser Val Asp 1 5 10 15 Val Val Ala Ala Leu Ile Arg MetAla Pro His Pro Cys Leu Leu Asn 20 25 30 Ile Val Ala Gln Thr Pro Leu HisLeu Ala Ala Leu Thr Ala Gln Pro 35 40 45 Asn Ile Met Arg Ile Leu Leu LeuAla Gly Ala Glu Pro Thr Val Arg 50 55 60 Asp Arg His Gly Asn Thr Ala LeuHis Leu Ser Cys Ile Ala Gly Glu 65 70 75 80 Lys Gln Cys Val Arg Ala LeuThr Glu Lys Phe Gly Ala Thr Glu Ile 85 90 95 His Glu Ala Asp Gly Glu ArgCys Val His Leu Ala Ala Glu Ala Gly 100 105 110 His Ile Asp Ile Leu ArgLeu Leu Val Ser His Gly Ala Asp Ile Asn 115 120 125 Ala Arg Glu Gly SerGly Arg Thr Pro Leu His Ile Ala Ile Glu Gly 130 135 140 Cys Asn Glu AspLeu Ala Asn Phe Leu Leu Asp Glu Cys Glu Lys Leu 145 150 155 160 Asn LeuGlu Thr Ala Ala Gly Leu Thr Ala Tyr Gln Phe Ala Cys Ile 165 170 175 MetAsn Lys Ser Arg Met Gln Asn Ile Leu Glu Lys Arg Gly Ala Glu 180 185 190Thr Val Thr Pro Pro Asp 195 7 165 PRT Arabidopsis sp. misc_feature(1)..(165) NPR1 protein 7 Ala Ala Val Lys Leu Glu Leu Lys Glu Ile AlaLys Asp Tyr Glu Val 1 5 10 15 Gly Phe Asp Ser Val Val Thr Val Leu AlaTyr Val Tyr Ser Ser Arg 20 25 30 Val Ile Pro Glu Leu Ile Thr Leu Tyr GlnArg His Leu Leu Asp Val 35 40 45 Val Asp Lys Val Val Ile Glu Asp Thr LeuVal Ile Leu Lys Leu Ala 50 55 60 Asn Ile Ile Val Lys Ser Asn Val Asp MetVal Ser Leu Glu Lys Ser 65 70 75 80 Leu Pro Glu Glu Leu Val Lys Glu IleIle Asp Arg Arg Lys Glu Leu 85 90 95 Gly Leu Glu Asp Ala Cys Ala Leu HisPhe Ala Val Ala Tyr Cys Asn 100 105 110 Val Lys Thr Ala Thr Asp Leu LeuLys Leu Asp Leu Ala Asp Val Asn 115 120 125 His Arg Asn Pro Arg Gly TyrThr Val Leu His Val Ala Ala Met Arg 130 135 140 Lys Glu Pro Gln Leu IleLeu Ser Leu Leu Glu Lys Gly Ala Ser Ala 145 150 155 160 Ser Glu Ala ThrLeu 165 8 1978 DNA Oryza sp. 8 gacccagaaa catttcgaaa gatcagccaggcaatggggc ctttaggcgg ccctgatttt 60 gctgaacctt ctggaactga aggaacagaggaagaaggtg aatatgaaga tgaatctatc 120 gtccatcaca ctgccagtgt cggtgatgatgaggtaaggg ggcagagtgc taagtagtac 180 agctaaggat ttgaaattat tacttcctccgtttcatatt ataacacttc ctagcattgc 240 ccacattcat atacatgtta atgaatctagacatatatgt gcgcctagat tcattaatat 300 ctatatgaat atgggcaatg ctagaaagtcttataacctg aaacggaggt agtattgata 360 ttactattta gtctcgagct tgagagtttgtatatgtttc tatgtcttgt tggtgtgtaa 420 tgtataattt actagagaag tgtccattcgtgtgtgtgtg tatggttata taatatcttc 480 aattacagta atatgcctct ccgttttggttttgctctga acaacatgta taggttttcg 540 cacaaattgt gatctcgatg gccttttctgtttcattgtc aattcagctt gcctttcttt 600 acaagtttaa gtcatctaat agggtctgaagaaagcttta gatggtggag cagacaaaga 660 cgaacaacac ttggagggca gaagggccttacactttgta tgtggatatg gggaggtatg 720 caagtctgct taactaaacc caatgacaattgaaacctgt gcaagtagaa aatgccgaat 780 aaatactact ccctccgttt cataatgtaagtcattctag catttttcat attcatattg 840 atgtttatga atctagaaag acatcaatatgaatgtggga aatgctagaa tgacttacat 900 tgtgaaacgg aagaagtact attacctatttgttgttatt gcaaatgaca aggttagcaa 960 ctataaaaac atctcgttgc gaatcctgtgcaaaacggat tgcatgtatg cgtgactagt 1020 cttcagaaaa ttgcatgtat gcaatgtgacagttcattat gcaaaacggt gaacctactg 1080 ttgccatcag tatccccgat actaattgaagttctcctaa tgttttcttt tttccttttt 1140 ggtaatcagc tagcgttgaa ttcagcttagttgggggcta actgtctttt tgcattctat 1200 gatgagtttt gacaaattta ttaattttatcttttttttt ttttgctttt aacacacttc 1260 aagatatttt tggtagatgg aaaggtgcagagcttgctgg tttactttgt tgaagctaaa 1320 actttgttag tttttctggg gcagttcattgatgataatc cagacctcac aggtcaacca 1380 acagtcctcg gtttcaaaaa aaaaaaaaaatcccacagta acctgtcccg ttgaacattg 1440 cacaaacttg tcagatctgg tgcacctctcgtctagctat aatagtatcg aactatgagt 1500 ttccataacc ccgctgtttg tataattgcagttggtgtgc aatgctagag cacaaaagtt 1560 aatgaacgac aaactacctt ttgattcattctcttgtgga tctagaatgt ggtgtgagac 1620 tttttttttg ggagctgcat ctgctccttgttcactgact aatcaggatt tgggttaaac 1680 ttttgttttt cagttgaagt gtgcccaagtacttcttgag gcgggtgctg cagtggatgc 1740 tttggacaag aacaagaaca ctccgctgcattacgccgct ggctatggta tgaaggggtg 1800 cgtggatctt ttgctgaaga acggagccgctgtgtaagtt aaacctgctc gctttgctag 1860 ttgcgatcac atcatttttt ttgcattatattatttgact gtctcgaatt gcatcgcagc 1920 accctcgaaa acatggatgg caagacgcccattgacgttg cgagctcaac accaggat 1978 9 2127 DNA Oryza sp. misc_feature(1)..(2127) cDNA 9 ggccacgcgt cgactagtac gggggggggg ggggggcgctctccctccac gagccatcgt 60 cgctgcacct cgcggtctcc gccgctctcc ctccacgagtcgccgccgcc gccagcactc 120 agagagagag agagacggaa tacggggaga gacgtagatatggatagggt ttggtcaagg 180 gtattttggt cattacgaaa aataattgca tttctttctttttaaaaaaa tgaaaactta 240 acagtgttaa aatcagggcc aaacggagtg ttcatttttaaaaagtaggg tcaaataagc 300 aaactagaaa aagtagggtc atattggtaa ttaagcttcaaaacagggtc aaataagcaa 360 ttacccctaa aaaaaaaaga acttgtcagg gcagatcataccatcatcac ccacagctcc 420 ttgtaaagga gtaaagaaac tgaaattgga agtttatcatatcatgttag ttttttttgg 480 ttcatatcct catcctgata agaaaaatat caaccttgatttggtgttat agtagtagtt 540 tcttatgacc gacattattt ttgtatttta gaatttgtttatgtgattgt cagctgatga 600 gctgataaaa tcgaattggg aattatttgg tgcgttggtcaaatccatct tcattcatag 660 tagttgcgtt ctaatccact ttgcaacctc aatttttcgcggaaaagatt tatagcattg 720 cagcttccct catatattgt aagaagaaaa ggtagaaaagaagcaaggaa tcagttcttt 780 tattcagctt ctttactagc cagttttatg ctttgttgtcaaagctggct acggtaccat 840 gcgatgcatg tttctgattt gatcaattct cttgcagatgagaaaaaaag ttcaaaacca 900 caaggatcat ccaatgatca tcaagggttt ctgccaggaggctctcctgc aaatactttt 960 gattttgctt ctttgcacag cttgctcaat gatccatctgtaaaggagat agcagatcag 1020 attgcaaagg accctgcgtt cacccagata gcggagcaggcactggaagg ccaaggagaa 1080 cagggcatgc ctgcaataga cccttacatt gaaacaatgcaaaagttcat ggaaagcccc 1140 cattttttta caatggcaga gcgtcttggg gatgctcttgtgaaggatcc tgcaatgtcc 1200 agtctgctgg aaaacttgac tagtccaatg cataatgcaaagatagaaga gcgtgtttct 1260 cgtatgaagg aagatccagc cgtgaaatca attatggctgagttagagac tggtgatcct 1320 gctgcactga taaagtactg gaatgaccca gaaacatttcgaaagatcag ccaggcaatg 1380 gggcctttag gcggccctga ttttgctgaa ccttctggaactgaaggaac agaggaagaa 1440 ggtgaatatg aagatgaatc tatcgtccat cacactgccagtgttggtga tgatgagggt 1500 ctgaagaagg ctttagatgg tggagcagac aaagacgaagaagacttgga gggcagaagg 1560 gccttacact ttgtatgtgg atatggggag ttgaagtgtgcccaagtact tcttgaggcg 1620 ggtgctgcag tggatgcttt ggacaagaac aagaacactccgctgcatta cgccgctggc 1680 tatggtatga aggggtgcgt ggatcttctg ctgaagaacggagccgctgt caccctcgaa 1740 aacatggatg gcaagacgcc cattgacgtt gcgaagctcaacaaccagga tgaggttctc 1800 aagttgctgg aaaaggatgc cttcctgtag atcgcctttgttattctcat gggcgcatga 1860 acagtttggc tccaggatca tcattcttta atttgcgtcgtttggtgccg ccattcatat 1920 ttctttgcta cccagtggca gttcataaga tacggtgaaggggctgccac acaactgctg 1980 tggttcacga tgacttgtgt accccagctt tgtttctcttgttttcatta gtgcaatcga 2040 gattgtgtat ccacattttc tttttttttt cagtattgcgcatatatgtc ttttcctttt 2100 ctgtgaaaaa aaaaaaaaaa aaaaaaa 2127 10 10 DNAArtificial Sequence misc_feature (1)..(10) OPF-1 primer 10 acggatcctg 10

1. A method of conferring disease resistance to a monocotyledonous plantcomprising: stably transforming into the genome of a monocotyledonousplant, a nucleic acid comprising SEQ ID No:2, whereby improved diseaseresistance relative to the untransformed plant is conferred.
 2. A methodof conferring rice blast resistance to rice comprising: stablytransforming into the genome of a rice plant, a nucleic acid comprisingSEQ ID NO:2, whereby improved disease resistance relative to theuntransformed plant is conferred.
 3. A method of conferring resistanceto disease in a monocotyledonous plant comprising stably integratinginto the genome of said plant a nucleic acid having a sequence whichcodes for a protein comprising the ankyrin motif consensus sequence. 4.The method according to claim 3 wherein said monocotyledonous plant isselected from the group consisting of rice, barley, corn, wheat, andasparagus.
 5. A method of conferring resistance to rice blast disease ina monocotyledonous plant comprising stably integrating into the genomeof said plant a nucleic acid having a sequence which codes for a proteincomprising the ankyrin motif consensus sequence.
 6. The method accordingto claim 5 wherein said monocotyledonous plant is selected from thegroup consisting of rice, barley, corn, wheat, and asparagus.
 7. Themethod according to claim 1, wherein said monocotyledonous plant is aplant selected from the group consisting of rice, barley, corn, wheat,and asparagus.
 8. A method according to claim 2, wherein saidmonocotyledonous plant is a plant selected from the group consisting ofrice, barley, corn, wheat, and asparagus.